1. Field of Invention
This invention concerns a novel and improved process for long-term preservation of the heart for transplantation. The preservation process comprise of perfusing the heart at a warm temperature with a first novel physiological solution containing pyruvate, under normal physiological conditions to remove blood, increase flow, and load the cells with pyruvate. A second cardioplegic solution containing pyruvate and alcohol is used to effect the vasolidation, the heart arrest and decrease of metabolism rate to a basal metabolic stage. The heart having a cannulated aorta and left ventricular chamber allowing diffusion of gases and media, is submerged and stored in the first solution for periods longer than 24 hours.
2. Related Disclosures
Organ transplantation, in particular heart transplantation has become an important tool in saving lives in patients with irreversibly diseased or damaged organs. With increasing incidence of the circulatory diseases in the populations, the heart transplantation is used more and more to preserve a life of otherwise healthy individuals with badly injured heart following the heart attack, myocardial infarction or other heart conditions. Consequently, a demand for organs suitable for transplantation has risen substantially.
There are primary requirements for the organ to be suitable for transplantation. First, the organ must be healthy. Second, it must be transplanted or transplantable in certain time in which it is possible to preserve its normal physiological function. Third, it must be immunologically acceptable to the organ recipient.
The first requirement can be only be met by the physician removing the organ from the donor's body. The third requirement is increasingly being made possible by improved understanding of immune mechanism and method for preventing organ rejection by the recipient's immune system. The pharmaceutical industry is constantly developing and designing new immunosuppressant drugs which allow the easier immunological matching of the donor and the recipient and prevent, as much as possible, the organ rejection by recipient. Drugs such as azathioprine, monoclonal antibody muromonab-CD3, cyclophosphamide, cyclosporine and other recently discovered drugs such as for example drug known as FK-506 which supposedly cuts the rejection rate 90% now allow the suppression of immune reactions for up to 6 months at which time the body of the recipient is able to substantially rebuild proteins in the transplanted organ with their own proteins thus making it more immunologically acceptable. Moreover, when the graft tissue becomes accommodated within recipient's body, it can be maintained with relatively small and reasonably well tolerated doses of immunosuppressive drugs.
Consequently, the only remaining obstacle for the successful transplantation of organs is the preservation of their anatomical and functional integrity, in particular the preservation of their normal function for any length of time. With geographical spread of possible donors over the whole world, the length of time longer than 24 hours for preservation is extremely important.
This is particularly true for organs having a continuous life preserving function and/or high metabolic rate with high energy and oxygen demands. The heart is one of the unique organs which has both.
The main causes leading to death of the myocardial cells are ischemia, edema, acidosis, calcium overload, and a loss of the electrical potential across the membrane. Consequently, these causes must be either eliminated or ameliorated to such degree that they do not cause the death of cells.
The primary function of the heart is its continuous pumping of blood through the blood circulation system. That function depends on uninterrupted myocardial contractility which, in turn, depends on uninterrupted supply of energy and oxygen. Myocardial contractility must be preserved even during the time when the heart is removed from the donor and transplanted into the recipient. Since the contracting heart needs the constant supply of energy and oxygen, if these are not available, myocardial ischemia, caused by inadequate circulation of blood to the myocardium develops, which in turn results the cell death and in the in irreversible destruction of the myocardial contractility.
Preservation of myocardial function from ischemic injury during cardiac arrest is currently commonly achieved by hypothermia and perfusion with certain cardioplegic solutions of which the most widely used cardioplegic agent is cold potassium chloride (15 to 35 mEg/1) solution. However, the potassium chloride is known to cause vasoconstriction and combined with hypothermia, such cardioplegic solution does not allow for full flow and washout of tissue. Ann. Chir. Gynecol., 76:22 (1987); Postgraduate Med. J., 59:11 (1983); Canad. Anaesth. Soc. J., 27:381 (1980); J. Suro. Res., 43:179 (1987).
Cardiac surgical procedures including a removal of the heart for transfusion and inserting the heart to the donor body often require a bloodless, relaxed and motionless field during operation. This is easily accomplished by ischemic arrest induced by cross clamping the aorta which accelerates ischemia and prevents exchange of gases. Any period of ischaemia accompanied therefore by oxygen deficiency causes metabolic and structural changes which determine the functional recovery of the heart in the postoperative period. The safe period of ischaemia for the human heart is not clearly defined but 20-30 minutes is generally considered to be the upper limit. When aortic cross clamping time exceeds this period, substantial subendocardial necrosis may occur, with low output syndrome in the postoperative period. The need for protection of the myocardium during ischemic arrest has been well recognized and a number of methods including local and systemic hypothermia, intermittent coronary perfusion, retrograde coronary perfusion with cold blood, coronary perfusion with cold lactated Ringer's solution, tetrodoxin, acetylcholine, chemical asanguinous K.sup.+ cardioplegia and cold blood cardioplegia, have all been used in experimental studies and clinical practice. Of these, hypothermia and pharmacological arrest with cold cardioplegic solutions have now gained wide acceptance in clinical practice, despite their causing vasoconstriction.
Hypothermia has been proved to be an effective method of myorcardia preservation. It provides a bloodless arrested heart, lowers the energy requirements, delays the depletion of high energy phosphate reserves and lactic acid accumulation, and retards the morphological and functional deterioration associated with ischemic arrest. The technique of topical cooling with continuous irrigation of the surface of the heart was first described in Surg. Gynaecol. Obst., 129:750 (1959). In this technique, the cooling proceeded from the surface of the heart to the interior and was unlikely to cool the subendocardium and the interventricular septum due to mediastinal and bronchial collateral return, which would warm the endocardial surface without profound hypothermia.
Moderate hypothermia and surface cooling have been generally found inadequate to protect the myocardium for more than one hour of ischemic arrest. On the other hand, deep hypothermia and surface cooling which have been sufficient of 90 minutes of ischemic arrest is known to cause myocardial damage due to crystallization of the membrane lipids and poor ventricular performance on perfusion. Canad. Anaesth. Soc. J., 27:381 (1980). Postgrad. Med. J., 59:11 (1983) reports that in addition to affording protection by reducing heart rate, hypothermia slows all metabolic processes (thus conserving energy) including damaging degradative mechanisms and pathways which produce toxic metabolites. The efficacy of hypothermia as a protective agent was reported as the post-ischemic recovery of function following a 60 minute period of ischemic arrest in the rat heart which is related to the degree of hypothermia during ischaemia. Reducing the myocardial temperature during ischaemia from 37.degree. C. to 4.degree. C. resulted in a progressive improvement of post-ischemic recovery from 0% to 96% of pre-ischemic function. The hypothermic protection is reported to be poor and falling off rapidly as the myocardial temperature rises above 28.degree. C. In contrast, below 24.degree. C. protection was excellent and was little improved with increasing degrees of hypothermia. The reason for the sharp inflection is unknown but might be related to lipoprotein phase transitions in cell membranes. If a similar relationship exists for the human heart, such results would strongly support the maintenance of myorcardial temperatures below 25.degree. C. during periods of ischemic arrest. Since however, hypothermia causes constriction of the vessels, the blood remains in the vessels and leads to clotting and blockage of vessels.
In recent years, there has been considerable controversy over the extent to which hypothermia and chemical arrest are additive. And in a recent series of studies (Ibid.) in the rat and the dog, these effect have been clearly demonstrated. Dog hearts were subjected to 120 minutes of ischaemia at 20.degree. C. In the hypothermia-alone group, non-cardioplegic solution was infused at 20.degree. C. at the onset and after 60 minutes of ischaemia. In the hypothermia-plus-cardioplegia group, the infusion conditions were identical, with the exception that a high potassium-containing protective solution was used. Measurements of ventricular function before and after bypass revealed significantly better recoveries in the hypothermia-plus-cardioplegia group than in the hypothermia-alone group. Recoveries of cardiac output, left ventricular minute work and dP/dt in the hypothermia-plus-cardioplegia group were 92%, 62% and 91% respectively, whereas in the hypothermia-alone group the values were 38%, 17% and 43% respectively.
While the above used combination of cardioplegia with hypothermia was able to recover the cardiac function to about 92%, the time of ischaemia was limited to 120 minutes with the upper possible period of ischemic arrest around 4 hours.
In view of the above findings that (a) the moderate hypothermia above is inadequate to protect the myocardium for more than 1 hour; (b) profound cooling of myocardium causes myocardial damage; and (c) that combination of cardioplegia and mild hypothermia can only preserve the myocardial function for up to around 4 hours, it is clear that the technique which would be able to avoid deep hypothermia and still be able to preserve around 90% of normal function of myocardium after 24 hours ischemia would be extremely advantageous.
The principles of successful cardioplegic protection have been outlined (Ibid. p.11) as follows: energy conservation through the chemical induction of rapid and complete diastolic arrest; slowing of metabolic rate and degradative processes through the coincident use of hypothermia; and the prevention or reversal of certain unfavorable ischaemia-induced changes with various protective agents.
One of the means which was previously used to achieve almost immediate reduction in myocardial contractile activity is by aortic cross clamping. This reduction is however not complete and a reduced level of contractile activity is maintained sometimes for several minutes before the onset of diastolic arrest. This activity may also recur intermittently during the ischemic period. In addition to delaying a hampering surgical activity, this mechanical activity wastes substantial amounts of cellular energy. During myocardial ischaemia, ATP and creatine phosphate production is severely restricted. Those supplies which are available are used in an attempt to maintain cellular homeostasis such as, for example, transmembrane ion gradients and tissue protection. Needless depletion of these limited energy reserves by ischemic contraction can only hasten the process of cell death.
Other attempts to achieve rapid diastolic arrest were made by using various chemical means. Cardioplegic solutions were investigated containing high concentrations of potassium where coronary infusion of a solution containing 16 mmol potassium chloride/liter causes complete arrest within a few seconds. The effect of this upon myorcardial energy reserves and resistance to ischemia has been investigated in a study in which isolated rat hearts were subjected to a 2 minute period of coronary infusion with a cardioplegic (16 mmol potassium/liter) or a non-cardioplegic (5 mmol potassium/liter) solution immediately following aortic cross clamping. After 30 minutes of ischemia, the cardioplegic hearts contained 11.1.+-.4.2 .mu.mol of ATP/g dry weight and 9.4.+-.2.1 .mu.mol of creatine phosphate/g dry weight, whereas the corresponding figures in the non-cardioplegic group were 5.3.+-.0.9 and 2.8.+-.0.4 .mu.mol/g dry weight respectively. This striking difference in high energy phosphates was reflected in the post-ischemic recovery of function, which was zero in the non-cardioplegic group as opposed to almost 50% in the cardioplegic group.
Potassium is not unique in its ability to induce cardiac arrest. Numerous other agents have been used clinically and/or experimentally, for example zero calcium, high magnesium, acetylcholine, neostigmine and tetrodotoxin. In each instance, the primary protective effect has been through rapid induction of arrest and conservation of cellular energy supplies. In the light of current knowledge, however, some agents such as zero calcium or tetrodotoxin could not be recommended for clinical use, or in case of transplantation of the heart.
Thus it would be desirable to be able to achieve the rapid diastolic arrest without using undesirable agents.
Successful preservation of the heart for transplantation depends on maintenance or restoration of the full myocardial contractility.
Numerous attempts have been made to improve the biochemical properties of cardioplegic solutions and to optimize myocardial protection during the extreme form of ischemia. This attempts are described in Biomed. Biochim. Acta., 46:499 (1987); Circulation. 76:180 (1987); J. Suro. Res., 42:247 (1987); Clin. Physiol., 7:43 (1987); and Cardiovasc. Surg., 91:259 (1986).
The most commonly used cardioplegia is crystalloid cardioplegia consisting of isotonic or slightly hypertonic saline supplemented with glucose and potassium chloride of which buffering capacity is usually afforded by the addition of sodium bicarbonate or THAM. In addition, some solutions contain small amounts of magnesium or calcium, glucose, ATP, creatine phosphate while others contain pharmacologic agents such as mannitol, insulin, procaine or calcium channel blockers. Blood cardioplegia is also investigated but was not better than the other cardioplegia.
Despite these advances in cardioplegia, a significant percentage of patients continue to demonstrate clinical evidence of myocardial damage in the postoperative period (New Engl. J. Med., 301:135 (1979), indicating that the current cardioplegia is not suitable for purposes of the heart preservation for transplantations for longer period of time.
For preservation of cellular mitochondrial function, it is important to arrest the heart immediately since significant utilization of high energy phosphates occurs during the brief period of contractile activity between the onset of ischemia and the onset of asystole. J. Thorac. Cardiovasc. Surg., 77:803 (1979); J. Surg. Res., 24:201 (1978). This is particularly important since myocardial recovery from prolonged global ischemic arrest depends in part on the conservation of high energy phosphate stores, on the presence of the substrate in the cardioplegic solution and on the avoidance of reperfusion injury at the cellular level. No substrate present, ischemia occurs, when the glucose is used as the substrate, edema develops. J. Mol. Cell. Cardiol., 13:941 (1981).
Decrease in cardiac performance due to insufficient supply of free energy is well documented. A reduction in contractile performance of isolated hamster heart correlates with a decrease in free energy of ATP hydrolysis. Cardiac Adaptation to Hemodynamic Overload, Training and Stress., 197 (1983) Ed. R. Jacob et al., Steinkopff Verlag.
When the glucose was used as a sole substrate edema developed and the high energy phosphates ATP and phosphocreatine reached maximum values during heart diastole and minimum during systole. Upon exhaustion of ATP, a decrease in high level phosphate accompanied by a low level in the free energy of ATP hydrolysis, augmented levels of lactate and inorganic phosphate resulted in a 50% reduction of cardiac performance. Cir. Res., 53:759 (1983).
Since during the myocardial contraction the high level energy is required, and since in particular in the heart removed from the donor for the transplantation the supply of nutrients is limited to those present in the cardioplegia, and since glucose which is currently used as the energy supply, metabolizes in the muscle cells to lactate, the myocardial tissue soon faces a metabolic acidosis. Also, there may be an incomplete breakdown of glucose in cells resulting in lower than 6 carbon sugar or phosphorylated sugar which may cause intracellular ionic inequilibrium which causes water to be retained intracellularly with edema. Under high work-load conditions with glucose as a sole substrate, glycolytic production of pyruvate is inadequate to meet the energy needs under aerobic or anaerobic conditions, and consequently the acidosis develops. The acidosis, particulary combined with cellular edema in turn may cause a number of detrimental effects on cardiac function during myocardial ischemia including electrophysiological abnormalities and a reduction in ventricular performance, gradual decrease in tension, and decrease in coronary flow. The metabolic depletion of ATP impairs the post ischemic recovery of myocardial performance.
If glycolysis is rate limiting, there is reduced delivery of pyruvate to the mitochondria. By substituting pyruvate for glucose glycolysis is bypassed and pyruvate is available to the mitochondria for oxidative phosphorylation producing free energy ATP. Ann. J. Physiol., 253: H 1261 (1987). The use of pyruvate as a sole exogenous substrate results in greater functional and biochemical recovery after 30 minutes of ischemia and 30 minutes of reflow.
Circ. Res., 35:448 (1974) reports that intracellular Ca.sup.2+ -overload, the major factor contributing to myocardial injury during global ischemia in reperfusion, leads to impaired oxidative phosphorylation, increased ATP breakdown and consequently inefficient ATP utilization for mechanical work. Many studies document the usefulness of a calcium antagonist as adjuncts to cardioplegia in order to prevent intracellular Ca.sup.2+ accumulation and its deleterious effects Ann. Thorac. Surg., 42:593 (1986), Am. Heart J., 118:1219 (1989).
Using the currently available cardioplegic solutions, the safe time of global myocardial ischemia in human hearts is limited to 120 minutes. As reported in Heart Disease, 1962 (1988), 3rd Ed., Harcourt Brace Jovanovich, within this period no significant myocardial necrosis or permanent functional damage results. Under these conditions, Principles in Surgery, 407 (1984) 4th Ed., McGraw Hill, suggest that hearts for transplantation in humans must be implanted within 4 hours from the time of the heart removal.
Therefore, it would be advantageous for a world-wide transplantation network to extend this time period to possibly 24 hours or more. With the transportation feasibility to connect around the world in 24 hours, a supply of the hearts for transplantation could be widely improved and made practical if these hearts were able to be fully functional after 24 hours.
It has been previously reported, that the heart is severely affected by ingestion of large amounts of alcohol. Changes such as impaired sodium, potassium stimulated ATPase activity, inhibition of sodium-calcium exchange, decreased fatty acid oxidation, depressed ATP, impairment of mitochondrial function and diminished ratios of phosphate to oxygen all lead to a reduction in cardiac contractility. While higher alcohol concentrations produce a sudden cardiac arrest in the isolated hamster and rat heart, acute alcohol exposure reversibly depresses cardiac function without affecting energy resources. FASEB. J., 2:256 (1988) and Mag. Res. in Med., 8:58 (1988) reported that perfusion of the isolated hamster heart with 2% ethanol for 30 minutes, showed decrease in developed pressure, a marked increase in end-diastolic pressure, a decrease in ATP and an increase in inorganic phosphate and prevented a cellular edema. There was no change in phosphocreatine or intracellular pH. After reequilibration, all the above values returned to almost normal levels showing that alcohol induced functional cardiac depression is reversible.
It is a primary object of this invention to provide an improved technology for long-time preservation of the heart for transplantation by using novel cardioplegic solutions and process of using these solutions to achieve the almost complete functional myocardial contractility restoration after 24 hours ischemia.